915
edits
Changes
changed beckman tube order number
*100% ethanol
*2 mm electroporation cuvette
*25 cm2 and 75 cm2 tissue culture flasks.
*Ultraclear pollyallomer tubes for Ti45 rotor (Beckman Cat# 344087)
*2x Virus storage buffer (10 mM Tris, pH 8.0, 100 mM NaCl, 0.1% BSA and 50% glycerol; filter sterilized)
==Protocol==
#Add 8 uL resuspended linearized plasmid to 20 uL electrocompetent cells
#Carefully transfer to ice cold 2 mm electroporation cuvette avoiding bubbles and keeping on ice
#Electroporate at '''pulse at 2,500 V, 200 O and 25 mF'''
#Resuspend in 500 uL LB and plate on 2-5 LB/Km plates
#Pick 10-20 of the smallest colonies the next day and grow in LB/Km broth
#Miniprep clones. Digest 10 uL with PacI and check size by running out both digested and undigested clones on a 0.8% agarose gel. Correct recombinants usually yield a large fragment (approximately 30 kb) and a smaller fragment of 3.0 or 4.5 kb.
#Retransform and amplify correct clone(s).
===Transformation into 293A Cells===
#Plate cells so that at time of transformation they are 50-70% confluent.
#Digest recombinant plasmid with PacI in 100 uL with 3ug plasmid and 100U PacI.
#Precipitate with 70% ethanol, dry and resuspend in 20 uL sterile water.
#Mix 3 ug PacI digested plasmid and 15 uL LipofectAMINE reagent for each 25-cm2 tissue culture flask in 500 uL Opti-MEM I, and incubate the DNA/LipofectAMINE reagent mix for 15–30 min at room temperature.
#While waiting, wash cells with serum free DMEM and add OptiMEM (5 mL/10 cm dish)
#Add DNA/Lipofectamine and incubate 4-6h.
#Replace media with complete DMEM
#The next day check transformation efficiency by GFP fluoresence (for pAdtrack)
#Do not remove media, but add 2 mL fresh complete DMEM every 5-7 days. Wait 2-3 weeks before collecting viral particles.
===Preparation and Purification of Viral Particles===
#Scrape cells into 15 mL conical tube.
#Aspirate all but the final 2 mL of cells and resuspend by vortexing
#Release viral particles with four freeze-thaw cycles (liquid nitrogen then 37C water bath)
#Centrifuge at 500g at 4C to pellet debris.
#Store supernatant (virus) at -80 or use immediately to amplify
===Amplification of Adenovirus===
#Plate 293A cells in 25-cm2 tissue culture flasks at 80–90% confluency in 7 ml complete DMEM 6–15 h before infection.
#Infect 293A cells by adding 40–50% of the primary transfection viral supernatants (i.e., 0.5–1.0 ml of the 2.0 ml viral lysate) to each 25-cm2 flask.
#Check transduction by GFP fluorsesence (for pAdtrack)
#Collect cells by scraping into a 15 mL conical 3-5 days post-infection.
#Remove all but 5 mL media by aspiration.
#Freeze/Thaw cells 4 times as described above
#Centrifuge at 500g to pellet debris
#Store supernatant (virus) at -80 or use immediately to amplify again.
#Repeat amplification three time more first with one 75 cm2 flask (using entire supernatant) then with 3-5 75 cm2 flasks, then with 10-20 75cm2 flasks using entire viral supernatant as the innoculant each time.
#For final amplification resuspend pelleted cells in 8 mL D-PBS -/-, perform 4 freeze thaw cycles and centrifuge 10 min at 7000g
===Purification of Adenovirus===
#Transfer supernatant to 50 mL conical tube and add 4.8g of Cesium chloride and dissolve
#Transfer 10 mL to 12 mL polyallomer tubes (for SW 45-Ti) add ~ 2mL mineral oil to top of tube to prevent tube crushing.
#Centrifuge 18–24 h at 176,000g (SW 41 Ti rotor at 32,000 r.p.m.)
#The purified virus should be 1-2cm below the mineral oil in an opaque layer.
#Remove virus layer to a clean falcon tube using a syringe (do this in a beaker to easier add bleach to waste).
#Add an equal volume of 2X Virus storage buffer and store at -80
#Titer the virus using a kit.
